Enzyme and Microbial Technology 52 (2013) 203–210
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Enzyme and Microbial Technology
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 ଝ
Novel sources of -glucanase for the enzymatic degradation of schizophyllan
a a,∗ a a
Nongnuch Sutivisedsak , Timothy D. Leathers , Kenneth M. Bischoff , Melinda S. Nunnally ,
b
Stephen W. Peterson
a
Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service,
U.S. Department of Agriculture 1815 North University Street, Peoria, IL 61604, USA
b
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service,
U.S. Department of Agriculture 1815 North University Street, Peoria, IL 61604, USA
a r t i c l e i n f o a b s t r a c t
Article history: Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a -1,3-linked
Received 29 October 2012 
backbone and -1,6-linked side chains of single glucose units at every other residue. Schizophyllan is
Received in revised form 3 December 2012
commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information
Accepted 4 December 2012
is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful
for controlled modifications of the polymer for novel applications. Enrichment cultures were used to
Keywords:
isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon
-glucanase
source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains
Hypocrea nigricans

showing the highest levels of -glucanase activity were identified as Penicillium simplicissimum, Penicil-
Penicillium simplicissimum
lium crustosum, and Hypocrea nigricans. -glucanases also showed activity against the similar -glucans,
Penicillium crustosum

Schizophyllum commune laminarin and curdlan. By comparison, commercial -glucanase from Trichoderma longibrachiatum and
Schizophyllan laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of

the novel isolates. -glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of
◦ ◦
60 C and 50 C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifi-

cations of -glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple
active endoglucanase species, including a 20–25 kD enzyme from P. simplicissimum.
Published by Elsevier Inc.
1. Introduction this assumption. However, surprisingly little information is avail-
able on the biodegradation of schizophyllan. S. commune has been
Schizophyllan is a polysaccharide produced by Schizophyllum reported to produce endo--1,3-glucanase [5], and Rau [2] pro-
commune, a white-rot fungus and ubiquitous mushroom. It is a posed that the organism can consume schizophyllan as a carbon
 
homoglucan with a -1,3-linked backbone and single -1,6-linked source, contributing to a loss of polysaccharide molecular weight
glucose side chains at every other residue [1,2]. Schizophyllan in late cultures. Lo et al. [6] described -glucosidases from S. com-
acts as a biological response modifier and a non-specific stimu- mune. Fontaine et al. [7] reported that schizophyllan was slightly
lator of the immune system. It is used in vaccines, anti-cancer hydrolyzed by one of two exo--1,3-glucanases associated with the
therapies, and as a bioactive cosmetics ingredient. Schizophyl- cell walls of Aspergillus fumigatus. On the other hand, Kanzawa et al.
lan can form oxygen-impermeable films for food preservation [3]. [8] found that exo--1,3-glucanase from Bacillus circulans rapidly
It also has been tested for use in enhanced petroleum recovery hydrolyzed curdlan and laminarin, but did not attack schizo-
[2,4]. phyllan. Tanji et al. [9] reported that schizophyllan was partially
As a natural polysaccharide, it can be assumed that schizo- degraded at a very slow rate in rats, to lower molecular weight
phyllan is biodegradable, and many of its applications rely on forms of <10,000 that were excreted in urine. It is potentially valu-
able to identify enzymes that attack schizophyllan, particularly for
use in controlled modifications of the polymer for novel applica-
tions.
In the current study, 23 novel strains were isolated that were
ଝ
Mention of any trade names or commercial products in this publication is solely capable of growing on schizophyllan as a sole carbon source. Strains
for the purpose of providing specific information and does not imply recommen-
showing the highest activities against schizophyllan were identi-
dation or endorsement by the U.S. Department of Agriculture. USDA is an equal
fied, and -glucanase activities were characterized. Results indicate
opportunity provider and employer.
∗ that novel fungal isolates are promising sources of schizophyllan-
Corresponding author. Tel.: +1 309 681 6620; fax: +1 309 681 6040.
E-mail address: [email protected] (T.D. Leathers). degrading enzymes.
0141-0229/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.enzmictec.2012.12.002
204 N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210
Table 1
containing 1.0% (w/v) commercial schizophyllan in a 50 mL flask with three 10 mm
a ◦
-glucanase activity against schizophyllan produced by newly isolated strains
glass beads. Cultures were incubated at 200 rpm for 7 days at 28 C, then centrifuged
×
 at 3220 g to produce cell-free culture supernatants. Mycelial pellets were dried at
Strain number Isolation sites -glucanase ◦
b 60 C for 48 h. All experiments were carried out in triplicate and standard deviations
(near Peoria, Illinois) (U activity/mL)
are shown.
1-1 Woodland 0.24 ± 0.02
1-2 0.16 ± 0.03
1-3 0.17 ± 0.03
2.4. Enzyme and protein assays
±
2-1 Prairie grassland 0.005 <0.001
Quantitative -glucanase assays were performed by the dinitrosalicylic acid
2-2 <0.001
(DNS) method [14] as modified by Leathers et al. [15]. Samples (5–20 L) were
2-3 0.003 ± <0.001
incubated in a total volume of 205 L containing 0.5% (w/v) substrate (schizo-
2-4 0.001 ± <0.001
 ◦
phyllan or another -glucan) in 50 mM sodium acetate buffer, pH 5.0, at 28 C.
3-1 Hayfield/woodland <0.001 Sample dilutions and incubation times were adjusted to ensure results were within
3-2 border <0.001 the linear range of the assays. One unit of enzyme activity is defined as the
amount of enzyme necessary to release 1 mole of glucose equivalents per min
±
4-1 Pond shore 0.007 <0.001
under the conditions tested. Schizophyllan (cosmetic grade) was purchased from
4-2 0.001 ± <0.001
European Technologies, Inc., Denver, CO. Other -glucan substrates (laminarin
± 6-1 Clay soil 0.008 <0.001 from Laminaria digitata, paramylon from Euglena gracilis, curdlan from Agrobac-

6-2 0.002 ± <0.001 terium sp. (Alcaligenes faecalis), and barley -glucan) were from Sigma-Aldrich, St.
6-3 0.21 ± 0.05 Louis. Commercial cellulases from Aspergillus niger and T. viride (Cellulysin) were

6-4 0.19 ± 0.01 from Calbiochem (La Jolla, CA). Commercial -glucanases from A. niger, T. lon-
gibrachiatum, and Bacillus subtilis, as well as laminarinase from Trichoderma sp.,
7-1 Marshland <0.001
were from Sigma–Aldrich. Temperature and pH optima were performed using the
7-2 0.08 ± 0.02
same assay. For studies of pH optima, substrate buffer was titrated to the desired
test pH with acetic acid or sodium hydroxide before digestion, then returned to
8-1 Backyard/gardening 0.007 ± <0.001
pH 5 before assays were developed, since the DNS assay is pH sensitive. Enzyme
8-2 area 0.003 ± <0.001
values are the mean of triplicate cultures and are characteristic of repeated exper-
8-3 0.003 ± <0.001
iments.
±
9-1 Laboratory contaminant 0.026 <0.001
Rapid, semi-quantitative -glucanase assays were performed using a solid
9-2 0.032 ± <0.001
medium plate assay. Samples (4 L) were spotted directly onto the surface of freshly
9-3 0.029 ± <0.001
prepared plates containing 0.7% (w/v) Phytagel (Sigma–Aldrich Co., St. Louis) and
a 0.05% (w/v) commercial schizophyllan in 50 mM sodium acetate buffer, pH 5.0.
Strains were isolated from enrichment cultures containing schizophyllan as a
Plates were incubated overnight and stained with an aqueous solution of 1 mg Congo
sole carbon source.
b red/mL for 45 min, then destained with 1.0 M NaCl for 30 min. Enzyme activity was
Pure cultures were grown for 7 days in basal medium containing 1.0% schizo-
observed as a clear zone. Laminarinase from Trichoderma sp. was used as a positive
phyllan as a sole carbon source.
control.
Extracellular protein was estimated by the Bradford method [16], with bovine
serum albumin as the standard.
2. Methods
2.1. Isolation of novel schizophyllan-degrading strains
2.5. Enzyme purification by fast protein liquid chromatography (FPLC)
Soil samples from the area of Peoria, Illinois were serially diluted according to
Leathers et al. [10]. Specifically, 2.0 g of soil was diluted into 198 mL of sterile 0.2%
Enzyme samples were purified using a fast protein liquid chromatography sys-
agar in distilled water (water agar). This was shaken vigorously, and then 10 mL was
tem (Biologic Duoflow, Bio-Rad, Hercules, CA). Cell-free culture supernatants were
transferred to 90 mL of water agar and mixed well. One milliliter of this suspension
applied to a 5.0 mL High Q anion exchange column (Bio-Rad) equilibrated in 50 mM
was then transferred into 9 mL of water containing 0.01% Tritron X-100. Aliquots
sodium acetate buffer, pH 5.0. Bound protein was eluted with an increasing gradi-
(0.1 mL) of these final dilutions were used to inoculate 10 mL enrichment cultures
ent of 0.0 to 1.0 M NaCl in the same buffer. Alternatively, samples were adjusted
containing 1.0% (w/v) schizophyllan (cosmetic grade, European Technologies, Inc.,
to 1.0 M (NH4)2SO4 by direct addition of solid (NH4)2SO4 and then applied to a
Denver, CO) as a sole carbon source in basal medium composed of 0.67% (w/v) yeast
×
Phenyl Sepharose column (1.5 10 cm, GE Healthcare, Piscataway, NJ) equilibrated
nitrogen base, 0.5% (w/v) KH2PO4, and 0.2% (w/v) bacto-asparagine (Difco Labora-
◦ with 1.0 M (NH4)2SO4 in 50 mM sodium acetate buffer, pH 5.0. Bound protein was
tories, Detroit). Enrichment cultures were grown for 7 days at 28 C and 200 rpm.
eluted with a decreasing gradient from 1.0 M to 0.0 M (NH4)2SO4 in the same
Serial dilutions were made onto solid medium containing potato dextrose agar (PDA,
buffer.
Difco), and isolates were single-colony purified at least three times. In addition, three
strains were isolated as laboratory contaminants of stored schizophyllan solutions
(Table 1).
2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
2.2. Sequence identification of strains
Active enzyme fractions from liquid chromatography were desalted using pro-
tein desalting spin columns (Pierce, Rockford, IL). Samples were denatured by
Conidia were floated free of the mycelium with 2–3 mL of 70% ethanol and
◦
heating for 4 min at 95 C in 2× SDS sample buffer (4.0% (w/v) SDS, 20% (v/v) glyc-
concentrated by brief centrifugation in a micro-centrifuge. Conidial pellets were
erol, 0.005% (w/v) bromophenol blue, 25% (w/v) 0.5 M Tris–HCl pH 6.8 and 5.0% (v/v)
suspended in 400 L of CTAB buffer [11] in a 1.5 mL microfuge tube containing
-mercaptoethanol) and applied to an SDS-PAGE gel (3.0% stacking, 10% resolv-
about 400 mg of 0.5 mm diameter glass beads. Cell walls were broken by vortex
ing). After electrophoresis at 100 V for approximately 1 h, the SDS PAGE gel was
mixing of the glass beads and conidia. Chloroform (0.4 mL) was added to extract
stained with SYPRO Ruby protein gel stain (Invitrogen, Grand Island, NY) for 16 h
proteins and the aqueous and organic phases were separated by centrifugation. The
and rinsed with deionized water. Stained gels were visualized by UV transillumina-
aqueous phase was transferred to a clean microfuge tube, precipitated by addition
tion.
of an equal volume of isopropanol and collected by centrifugation. The resulting
pellet was rehydrated in 100 L buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0). The DNA
preparation was diluted 10 to 100 times in sterile distilled water for use in PCR
amplifications. Beta tubulin was amplified using the primers and procedures of 2.7. Zymogram analysis
Glass and Donaldson [12]. Alternatively, DNA isolation was carried out using a ZR
Fungal/Baterial DNA kit following the procedure according to Nawrot et al. [13], and Samples from cell-free culture supernatants and liquid chromatography frac-
strains were identified by the sequence of their 28s rRNA genes. Primers were NL- tions were denatured as described above and applied to a 10% SDS-PAGE gel
1 (59-GCATATCAATAAGCGGAGGAAAAG) and NL-4 (59-GGTCCGTGTTTCAAGACGG) containing 0.04% (w/v) commercial schizophyllan within the gel matrix. Following
as described by O’Donnell [11]. electrophoresis, the gel was incubated in 50 mM sodium acetate buffer, pH 5.0 at
◦
28 C for 20 h, stained with an aqueous solution of 1.0 mg Congo Red/mL for 30 min,
2.3. Culture conditions for ˇ-glucanase production on schizophyllan and destained with 1.0 M aqueous NaCl for 30 min. After rinsing with deionized
water, enzyme activity was visualized on the gel as clear bands against a red back-
◦
Strains were grown on PDA slants at 28 C for 7–10 days. An approximately ground of stained schizophyllan. The clear band resulted from the degradation of
×
7 mm 7 mm square of mycelium was used to inoculate 10 mL of CB basal medium schizophyllan.
N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210 205
Table 2
Identification of novel strains producing -glucanase activity against schizophyllan.
a
Isolate number Strain number Species designation Match Colonial morphology
1-1 NRRL 62550 Penicillium simplicissimum 566/569 Low velutinous, conidia celadine green becoming olive centrally,
abundant clear exudates, reverse cream
1-2 NRRL 62551 Hypocrea nigricans 565/565 Rapidly spreading dark blue-green, numerous scattered cleistothecia
1-3 NRRL 62552 H. nigricans 558/558 Rapidly spreading dark blue-green, numerous scattered cleistothecia
6-1 NRRL 62553 Aspergillus flavus 566/566 Velutinous pea-green, clear exudates, numerous sclerotia, reverse
creamy
6-2 NRRL 62554 A. alliaceus 566/566 Floccose, clear exudates, central stromata
6-3 NRRL 62555 H. nigricans 558/558 Rapidly spreading dark blue-green, numerous scattered cleistothecia
6-4 NRRL 62556 H. nigricans 560/560 Rapidly spreading dark blue-green, numerous scattered cleistothecia
9-1 NRRL 62557 Penicillium crustosum 564/564 Low spreading velutinous, artemesia green, reverse pale yellow-orange
9-2 NRRL 62558 P. crustosum 564/564 Low spreading velutinous, artemesia green, reverse pale yellow-orange
9-3 NRRL 62559 P. crustosum 578/579 Low spreading velutinous, artemesia green, reverse pale yellow-orange
a
Sequence match to proposed species designations in GenBank database.
3. Results and discussion on this medium, and 20 isolates were purified from these cul-
tures by repeated single-colony isolations (Table 1). In addition,
3.1. Isolation of novel organisms capable of growth on three organisms were isolated as laboratory contaminants of stored
schizophyllan as a sole carbon source schizophyllan solutions.
Purified strains were cultured in basal medium containing
Since schizophyllan is produced by the ubiquitous mushroom, schizophyllan as a sole carbon source. All cultures exhibited
S. commune, novel organisms capable of degrading schizophyl- good growth on schizophyllan. Mycelial dry weights ranged from
lan were sought in soil samples from 7 diverse environments 25–100 mg from cultures initially containing 100 mg schizophyllan
near Peoria, IL (Table 1). Soil dilutions were used to inoculate (data not shown). Cell-free culture supernatants were assayed for

enrichment cultures containing commercial schizophyllan as a -glucanase activity against commercial schizophyllan (Table 1).
sole carbon source. All soil samples tested provided good growth Five of 20 soil isolates exhibited -glucanase activity of approxi-
mately 0.2 U/mL (Table 1), while the three laboratory contaminants
showed activities of about 0.03 U/mL. The remainder of the iso-
120
A 
Strain NRRL 62550 lates showed lower -glucanase activities, some below the limits
Strain NRRL 62555
of detection by this assay (<0.001 U/mL). The 8 best producers of 100

-glucanase activities against schizophyllan were chosen for fur-
ther study. Low producer isolates 6–1 and 6–2 were included as 80
negative controls.
60
3.2. Identification of novel strains producing ˇ-glucanase activity
40 against schizophyllan
20 Ten novel isolates producing -glucanase activity against
Glucanase (% maximum)
schizophyllan were identified by sequence analysis and colonial 0
020406080
o
Tempe rature ( C)
B 120 Strain NRRL 62550 100 Strain NRRL 62555
80
+ - +
2 4 6 8 32 34
60
2 4 6 8 40 42 10 10 48 40
20 Glucanase (% maximum)
0
3 4 5 6 Fig. 2. SDS PAGE of cell-free culture supernatants. (A) BioRad Precision Plus pro-
tein standards; (B) commercial laminarinase from Trichoderma sp. (dil 10× from
pH
12 U/mL); (C–E) replicate cultures of Schizophyllan commune ATCC 38548; (F–H)
replicate cultures of Penicillium crustosum strain NRRL 62557; (I–K) replicate cul-

Fig. 1. Temperature (A) and pH (B) optima of -glucanases from Penicillium simpli- tures of P. crustosum strain NRRL 62558; (L–N) replicate cultures of P. crustosum
cissimum strain NRRL 62550 and Hypocrea nigricans strain NRRL 62555. strain NRRL 62559.
206 N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210
Table 3
Specificity of -glucanase activities produced by novel isolates grown on schizophyllan as a sole carbon source.

-glucanase activity (IU/mL) against substrates:
a
Strain number Schizophyllan Laminarin Paramylon Curdlan Barley -glucan Protein (mg/mL)
1-1 0.24 ± 0.02 0.60 ± 0.12 0.011 ± <0.001 0.23 ± 0.01 0.98 ± 0.13 0.089 ± 0.01
±
1-2 0.16 ± 0.03 0.34 0.12 0.004 ± <0.001 0.34 ± 0.04 0.60 ± 0.05 0.082 ± 0.01
1-3 0.17 ± 0.03 0.63 ± 0.04 0.005 ± <0.001 0.24 ± 0.02 0.68 ± 0.02 0.087 ± 0.00
6-1 0.008 ± <0.001 0.10 ± 0.03 0.002 ± <0.001 0.011 ± <0.001 0.036 ± <0.001 0.094 ± 0.00
6-2 0.002 ± <0.001 0.002 ± <0.001 0.001 ± <0.001 0.002 ± <0.001 0.02 ± 0.01 <0.01
6-3 0.21 ± 0.05 0.57 ± 0.04 0.005 ± <0.001 0.40 ± 0.02 0.73 ± 0.13 0.097 ± 0.01
6-4 0.19 ± 0.01 0.47 ± 0.07 0.005 ± <0.001 0.34 ± 0.02 0.62 ± 0.01 0.074 ± 0.01
±
9-1 0.026 ± <0.001 0.24 0.03 0.013 ± <0.001 0.029 ± <0.001 0.32 ± 0.05 0.078 ± 0.02
±
9-2 0.032 <0.001 0.16 ± 0.07 0.004 ± <0.001 0.037 ± 0.01 0.34 ± 0.03 0.081 ± 0.01
9-3 0.029 ± <0.001 0.19 ± 0.01 0.010 ± <0.001 0.034 ± <0.001 0.24 ± 0.03 0.052 ± 0.01
ATCC 38548 0.003 ± <0.001 0.10 ± 0.04 <0.001 0.013 ± <0.001 0.26 ± 0.01 0.050 ± 0.04
a
Activities against schizophyllan from Table 1.

Fig. 3. Partial purification of -glucanase from Penicillium crustosum strain NRRL 62558. (A) High Q anion-exchange column; (B) Rapid, semi-quantitative -glucanase assays
of High Q fractions (+ = laminarinase control, − = water control).
N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210 207
Table 4

Activity of commercial -glucanases against schizophyllan and other -glucans.

-glucanase activity (IU/mL) against substrates
Enzyme Schizophyllan Laminarin Paramylon Curdlan Barley -glucan Protein (mg/mL)
Cellulase, Aspergillus niger <0.001 <0.001 0.47 ± 0.01 0.50 ± 0.06 5.4 ± 0.01 0.05 ± <0.001
Cellulase, Trichoderma viride <0.001 <0.001 0.07 ± <0.001 0.24 ± 0.08 3.8 ± 0.01 0.18 ± <0.001

-glucanase, A. niger <0.001 <0.001 0.09 ± 0.01 0.35 ± 0.01 30 ± <0.001 3.5 ± 0.02
-glucanase, T.longibrachiatum 0.15 ± 0.01 <0.001 0.05 ± <0.001 0.45 ± 0.03 63 ± 0.03 1.9 ± <0.001
-glucanase,
Bacillus subtilis <0.001 <0.001 <0.001 <0.001 58 ± 0.01 1.2 ± <0.001
Laminarinase,
± ±
Trichoderma sp. 4.7 0.02 46 0.01 0.70 ± 0.02 19 ± 0.02 200 ± 0.01 11 ± 0.01
morphologies (Table 2). One isolate from a woodland isolation Penicillium crustosum (Table 2). Thus, most novel isolates produc-
site was identified as a Penicillium simplicissimum, while two were ing -glucanase activity against schizophyllan appeared to belong
shown to be Hypocrea nigricans. Two isolates from clay soil were to either Penicillium species or H. nigricans. These genera are well
identified as Aspergillus flavus and A. alliaceus, while the rest known for production of numerous hydrolytic enzymes, includ-


were H. nigricans. All three strains isolated as laboratory con- ing -glucanases known to be active against -glucans other than
taminants of stored schizophyllan solutions were identified as schizophyllan.

Fig. 4. Partial purification of -glucanase from Penicillium crustosum strain NRRL 62558. (A) Phenyl Sepharose column; (B) Rapid, semi-quantitative -glucanase assays of
−
phenyl sepharose fractions (+ = laminarinase control, = water control).
208 N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210
3.3. Specificity of ˇ-glucanase activities produced by novel
isolates
The specificity of -glucanase activities produced by novel iso-
lates grown on schizophyllan as a sole carbon source was tested in
assays on other -glucans, specifically curdlan, laminarin, paramy-
lon, and barley -glucan (Table 3). Enzymes were also tested from
S. commune strain ATCC 38548 cultured on schizophyllan. S. com-
mune has been reported to produce cellulase [17] and -glucanase
with activity against schizophyllan [5], and it is thought that the
organism can consume the polysaccharide as a carbon source in
late cultures [2]. While schizophyllan possesses a -(1,3) back-
bone with regular -(1,6) branches of one subunit [2], curdlan is
a water-insoluble -glucan produced by Agrobacterium sp. (Alca-
ligenes faecalis), composed primarily of linear -(1,3) linkages [18].
Laminarin, from the brown alga Laminaria digitata, is a water sol-
uble, mainly linear -(1,3) glucan with a small number of -(1,6)
Fig. 5. SDS PAGE gel of partially purified -glucanase fractions. (A) BioRad Precision
linked side chains [19]. Paramylon is similar to laminarin, but has
Plus protein standards; (B) commercial laminarinase from Trichoderma sp. (dil 10×

a much higher level of crystallinity [20]. Barley -glucan is a linear from 12 U/mL); (C) Penicillium crustosum strain NRRL 62558 total cell-free culture
supernatant; (D) P. crustosum strain NRRL 62558 Phenyl Sepharose column Area 1
homoglucan, mostly composed of two or three consecutive -(1,4)
(fraction 32); (E) P. crustosum strain NRRL 62558 High Q column Area 3 (fraction
linkages separated by a single -(1,3) linkage [21].
 32); (F) P. crustosum strain NRRL 62558 High Q column Area 2 (combined fractions
-glucanase activities from novel isolates were generally 2–10
16–24); (G) P. simplicissimum strain NRRL 62550 total cell-free culture supernatant;
times higher on laminarin than on schizophyllan (Table 3). Since (H) P. simplicissimum strain NRRL 62550 High Q column fraction 34.
 
laminarin is a water-soluble -(1,3)-glucan with far fewer -(1,6)
side chains than schizophyllan, this result suggests that the novel
paramylon. Fungal -glucanases also showed activity against curd-
isolates produce primarily -(1,3)-endoglucanases that attack the
lan and paramylon, although -glucanase from B. subtilis did not.
backbone of schizophyllan, and that the abundant side chains of
Results suggest that different commercial enzyme preparations
schizophyllan limit access to this backbone. However, activities
vary in enzyme composition and specificity.
against paramylon were uniformly lower than on schizophyllan.
Among commercial enzyme preparations tested, only -
Although the structure of paramylon is similar to that of lami-
glucanase from T. longibrachiatum and laminarinase from Tricho-
narin, its higher degree of crystallinity may also limit access to
derma sp. exhibited activity against schizophyllan (Table 4). These
enzymes. Activities against curdlan were equivalent to double
enzymes were 3–4 times more active against curdlan than against
those on schizophyllan, depending on the strain used. Curdlan
schizophyllan (Table 4). Curdlan may be a more accessible sub-
completely lacks side chains, and the polymer is consequently
strate than schizophyllan, due to its lack of side chains. However,
insoluble. This may explain activities intermediate between those
only laminarinase from Trichoderma sp. also exhibited activity
of schizophyllan and laminarin. Interestingly, enzymes from novel
against laminarin. Specific activities against schizophyllan were
isolates were generally more active against barley -glucan than

0.1 U and 0.4 U/mg protein for -glucanase from T. longibrachia-
against laminarin (Table 3). Since barley -glucan does not contain
tum and laminarinase from Trichoderma sp., respectively. These
consecutive -(1,3) linkages, this could mean that either -(1,3)-
activities are similar to those from novel P. crustosum strains
endoglucanases are able to recognize single linkages, or that novel
NRRL 62557, NRRL 62558, and NRRL 62559, but lower than spe-
isolates also produce -(1,4)-endoglucanases that attack the -
cific activities from novel P. simplicissimum strain NRRL 62550
(1,4) linkages in barley -glucan. Under conditions tested here, S.
and novel H. nigricans strains NRRL 62551, NRRL 62552, NRRL
commune produced only marginal activity against schizophyllan.
62555, and NRRL 62556, which ranged from 2.0 to 2.7 U/mg pro-
tein.
3.4. Activity of commercial ˇ-glucanases against schizophyllan
ˇ and other -glucans
ˇ
3.5. pH and temperature optima of -glucanase produced by
novel isolates
To further test the action of endoglucanases against schizophyl-
lan, a set of commercial enzymes was tested against the same
pH and temperature optima were determined for -glucanases
substrates. Cellulases from A. niger and T. viride hydrolyze the -
from P. simplicissimum strain NRRL 62550 and H. nigricans strain
(1,4) linkages in cellulose (Calbiochem). -glucanases from A. niger
NRRL 62555. Cell-free culture supernatants from strains NRRL
and B. subtilis degrade -1,4-glucans of cellulose and possibly other
62550 and NRRL 62555 exhibited rather sharp temperature optima
polymers (Sigma–Aldrich). -glucanase from T. longibrachiatum is
◦ ◦
at about 60 C and 50 C, respectively (Fig. 1A). Both strains showed
a mixture of enzymes composed mainly of -(1,3)/-(1,4) glu-

a broad pH optimum centered at about pH 5.0 (Fig. 1B). Thus, -
canase, xylanase, and cellulase (Sigma–Aldrich). Laminarinase from
glucanases from novel isolates appear to be relatively thermophilic.
Trichoderma sp. is defined by its activity on laminarin. However,
it is possible that all of these commercial preparations include
ˇ
multiple enzyme activities. For comparison purposes, all com- 3.6. Partial purification of -glucanase from P. crustosum
mercial enzymes were prepared at or diluted to12 U activity/mL,

based on advertised activities, in 50 mM sodium acetate buffer, -glucanase activities were partially purified from P. crustosum

pH 5.0. Assays were then run on schizophyllan and other - strain NRRL 62558. Cell-free culture supernatants from P. crusto-
glucans. sum strains NRRL 62557, NRRL 62558, and NRRL 62559 showed
All commercial enzymes tested were most active against barley similar protein patterns on SDS-PAGE, and quite different from

-glucan (Table 4). Cellulases also exhibited activity against curd- those from S. commune and commercial laminarinase (Fig. 2). All of
lan and paramylon. Interestingly, cellulase from A. niger was equally these samples produced clear zones when spotted on schizophyl-
active against these substrates, despite the higher crystallinity of lan substrate plates, indicating that they contained endo-glucanase
N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210 209

Fig. 6. Partial purification of -glucanase from P. simplicissimum strain NRRL 62550. (A) High Q anion-exchange column; (B) Rapid, semi-quantitative -glucanase assays of
High Q fractions. (+ = laminarinase control, − = water control, S = culture supernatant control).
activities against this substrate (data not shown). Culture super-
natant from strain NRRL 62558 was subjected to chromatography
using a High Q anion exchange column (Fig. 3A). -glucanase activ-
ity was measured by spotting column fractions on a schizophyllan
substrate plate (Fig. 3B). Three active fractions were identified:
High Q Area 1 included fractions 2–10, representing a broad pro-
tein peak; High Q Area 2 included fractions 16–22, representing a
large distinct protein peak; and High Q Area 3 included fractions
28–32, representing a small but distinct protein peak. High Q Area
1 was further purified on a Phenyl Sepharose column (Fig. 4). -
glucanase activity was associated with fraction 29–36, represented
by a small distinct protein peak (Phenyl Sepharose Area 1). Active
fractions from each of the partially purified peaks were combined,
desalted, and subjected to SDS-PAGE (Fig. 5). Phenyl Sepharose Area
1 (lane D) was enriched in protein species of approximately 30 kD
and 100 kD, also apparent in unfractionated culture supernatants
(lane C). On the other hand, High Q Areas 2 and 3 (lanes F and
E, respectively) were enriched in protein species of about 40 kD
and 75 kD, not sufficiently abundant to be evident in culture super-

natants. Thus, -glucanase activity in P. crustosum appears to be
associated with multiple protein species. Although little is known
about enzymes that specifically attack schizophyllan, a survey of
160 fungi found that fungal laminarinases often have multiple com-
ponents [22].
Fig. 7. Zymogram of -glucanase activities in SDS PAGE gel of partially purified ˇ
3.7. Partial purification of -glucanase from P. simplicissimum
-glucanase from P. simplicissimum strain NRRL 62550. (A) BioRad Precision Plus
protein standards; (B) commercial laminarinase from Trichoderma sp. (dil 10× from
Cell free culture supernatant from P. simplicissimum strain NRRL
12 U/mL); (G) Penicillium simplicissimum strain NRRL 62550 total cell-free culture
supernatant; (H) P. simplicissimum strain NRRL 62550 High Q column fraction 34. 62550 was subjected to chromatography using a High Q anion
210 N. Sutivisedsak et al. / Enzyme and Microbial Technology 52 (2013) 203–210
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